Tumor invasion and metastasis still claim the majority of cancer patients' lives. Although great strides have been made in cancer detection and prevention, the need for a more effective treatment for cancer metastasis remains a central problem. Notably, molecules that control spatial signaling for cell polarization and directed migration contribute to the lethality of cancer through their ability to improve the efficiency of he first steps of metastasis. RhoA contributes to cell polarization during directed cell motility, yet how RhoA is spatially controlled remains unclear. We propose that integrin signaling through cAMP/PKA is instrumental for spatial signaling of RhoA and cell polarization. Importantly, our previous work has shown that ?1 integrins control the generation of PKA activity gradients at the leading edge of migrating carcinoma cells where it controls RhoA function. We provide compelling evidence that these PKA activity gradients exist in breast carcinoma patient samples and in three-dimensional breast carcinoma cultures, which attest to the significance of these gradients in human breast cancer. Integrin??6?4 is upregulated in advanced breast cancers where it is most closely associated with basal-like breast cancers. Importantly, integrin ??6?4 promotes the activation of RhoA by a mechanism that we propose reverses the effect of PKA. Therefore, we suggest that the counter-opposing modes of regulation these integrins have on RhoA permit efficient compartmentalization of RhoA that facilitates breast carcinoma motility, invasion and metastasis. Accordingly, the central hypothesis of this application is that cooperative signaling between ?1 integrins and integrin ?6?4 facilitates the spatial distribution of RhoA activity to promote lamellae formation and directed migration. The long-term goal of our group is to determine how integrins contribute to tumor invasion so that they may be eventually targeted appropriately for therapeutic intervention, with special emphasis on integrin ?6?4. We will test our central hypothesis and achieve our long- term goal through the completion of the following aims: 1) Define the macromolecular complex that couples ?1 integrins to PKA activation thereby limiting RhoA activity at the leading edge, 2) Determine how integrin ?6?4 leads to the activation of RhoA, and 3) Elucidate how RhoA function is altered to promote lamellae formation. We are uniquely suited to perform these studies due to our strong collaborations; our well-characterized models of ?6?4 integrin- and RhoA-dependent invasion and migration; our expertise in integrin biology and Rho signaling, and our solid preliminary data supporting this project. The results obtained from this study will be important as they will go int mechanistic depth regarding how integrins coordinate spatial signaling to achieve polarization, lamellae formation, directed cell migration and, finally, invasion. Ultimately, our study will be significant as it will generate a firmer understanding of signaling pathways that govern leading edge dynamics that will produce successful methods to therapeutically target the early steps in metastasis. PUBLIC HEALTH RELEVANCE: Understanding how integrins signal continues to have a sustained impact on our understanding of cancer biology. Notably, integrins contribute to the most deadly aspects of cancer progression including tumor invasion and metastasis; yet how they contribute to these processes remains poorly understood. Here, we will go into mechanistic depth regarding how integrins coordinate spatial signaling to achieve polarization, lamellae formation, directed cell migration and, ultimately, invasion. By understanding leading edge dynamics of invading cells, better methods of therapeutically targeting the early steps in metastasis will be achievable.